Nonlinear ultrasound imaging of nanoscale acoustic biomolecules

Abstract

Ultrasound imaging is widely used to probe the mechanical structure of tissues and visualize blood flow. However, the ability of ultrasound to observe specific molecular and cellular signals is limited. Recently, a unique class of gas-filled protein nanostructures called gas vesicles (GVs) was introduced as nanoscale (∼250 nm) contrast agents for ultrasound, accompanied by the possibilities of genetic engineering, imaging of targets outside the vasculature and monitoring of cellular signals such as gene expression. These possibilities would be aided by methods to discriminate GV-generated ultrasound signals from anatomical background. Here, we show that the nonlinear response of engineered GVs to acoustic pressure enables selective imaging of these nanostructures using a tailored amplitude modulation strategy. Finite element modeling predicted a strongly nonlinear mechanical deformation and acoustic response to ultrasound in engineered GVs. This response was confirmed with ultrasound measurements in the range of 10 to 25 MHz. An amplitude modulation pulse sequence based on this nonlinear response allows engineered GVs to be distinguished from linear scatterers and other GV types with a contrast ratio greater than 11.5 dB. We demonstrate the effectiveness of this nonlinear imaging strategy in vitro, in cellulo, and in vivo.

FIG. 1.

Simulation of the hGV shell response to incoming ultrasound imaging pulses. (a) Diagram and finite element simulation of hGV shell deformation at 190 kPa and (b) at 200 kPa peak positive incident pressures (p_i). Gray lines represent an incident excitation wave, while black circles represent scattered wave. (c) Simulated radial excursion at 190 kPa normalized to the resting radius R₀ (top) and corresponding Fourier transform (bottom). (d) Simulated radial excursion at 200 kPa normalized to the resting radius R₀ and corresponding Fourier transform.

FIG. 2.

Experimental spectra of backscattered signals from PS, wt GVs, and hGVs. (a) PS spectra, (b) wtGVs spectra and (c) hGVs spectra in response to varying peak positive incident pressures. (d) Fundamental area under the curve (AUC) integrated from 9.5 MHz to 13 MHz as a function of pressure (N = 5 samples; error bars represent standard error of the mean). (e) Second harmonic AUC integrated from 19 MHz to 26 MHz as a function of pressure (N = 5 samples; error bars represent standard error of the mean). (f) Differential AUC response between hGVs and wtGVs at the fundamental (dashed red line) and second harmonic frequency (dashed blue line).

FIG. 3.

Simulation of the scattered frequency spectrum of a single hGV in response to an amplitude modulation and pulse inversion sequence. (a) Simulated spectrum from an amplitude modulation pulse sequence comprising one full-amplitude (380 kPa peak positive) and two subtracted half-amplitude (190 kPa peak positive) 6-cycle sine-bursts at 11.4 MHz. (b) Simulated spectrum form a pulse inversion sequence comprising the sum of two phase-inverted 6-cycle sine-bursts at 380 kPa peak positive amplitude and 11.4 MHz. The frequency spectra are normalized Fourier transforms of the scattered pressure computed from changes in the simulated effective radius of the GVs.

FIG. 4.

In vitro nonlinear imaging of hGVs versus PS and wtGVs. (a) Conventional ultrasound B-Mode imaging acquired using 11.4 MHz 6-cycle sine-bursts. Left, phantom image comparing PS to hGVs. Right, phantom image comparing wtGVs to hGVs. (b) Amplitude modulation pulse sequence consisting of the sequential transmission of one full amplitude and two half-amplitude 11.4 MHz 6-cycle sine bursts. Left, phantom image comparing PS to hGVs. Right, phantom image comparing wtGVs to hGVs. (c) Pulse inversion sequence consisting of the sequential transmission of two phase inverted 11.4 MHz 6-cycle sine bursts. Left, phantom image comparing PS to hGVs. Right, phantom image comparing wtGVs to hGVs. (d) Ratios of hGV to PS contrast at 11.4 MHz for B-Mode, amplitude modulation and pulse inversion imaging (N = 5 samples; error bars represent standard error of the mean). (e) Amplitude modulation images at 18 MHz. Left, PS versus hGVs. Right, wtGVs to hGVs. (f) Selective amplitude modulation imaging of hGVs embedded within a phantom filled with PS. Scale bars represent 1 mm. PS and wtGV inclusions were imaged at a depth of 8 mm.

FIG. 5.

In cellulo and in vivo nonlinear imaging of hGVs at 18 MHz. (a)–(d) In cellulo images of hGVs in Xenopus laevis oocytes. (a) B-Mode imaging of 5 oocytes. The first three oocytes, labelled with white arrows, were injected with hGVs (50 nl, 1.8 nM). (b) Corresponding amplitude modulation image. (c) and (d) Images of the same sample after collapsing hGVs. (e)–(h) In vivo imaging of a wild-type mouse after hGVs were introduced into its colon. Left, B-Mode images before (top) and after (bottom) collapse. Right, AM images before (top) and after (bottom) collapse. Scale bars represent 1 mm. Oocytes and hGVs were imaged at a depth of 8 mm.